The active sites of Escherichia coli galactose-1-P uridylyltransferase and UDP-galactose 4-epimerase will be characterized with the objective of identifying amino acid functional groups involved in catalysis or binding of substrates. First priority will be given to developing a new technique for labeling and identifying enzymic groups at nucleotide binding sites. Sulfur analogs of nucleotides bound at active sites will be activated by reaction with electrophilic reagents to convert the sulfur nucleotides into active phosphorylating agents for phosphorylating active site residues. The 32P-labeled protein will be degraded to phospho amino acids to identify the phosphorylation sites. This approach will be complemented by the use of group-selective reagents and active site-directed alkylating agents to modify other functional groups. The catalytic functions of any active site-residues identified will be further investigated in studies of the reaction kinetics. The pH-dependence of V, Km and V/Km using both normal and slowly reacting substrates will be determined. It is expected that with slowly reacting substrates product dissociation step will not limit rates, and under these conditions the pH-rate data may give valuable information about the pKa values for catalytic groups in the active sites of these enzymes. Isotope effects and exchange rates will be measured to determine whether product dissociation steps are or are not partially rate limiting for slow substrates.